The lysosome is an organelle founded in the cytoplasm of eukaryotic cells, which serves as storage for many hydrolytic enzymes and as a center for degrading and recycling cellular components. This organelle contains several types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases and sulfatases. All enzymes are acid hydrolases. See Lehninger et als, Principles of Biochemistry, 2nd ed., Worth Publishers, Inc., New York (1992).
Lysosomal storage diseases (“LSDs”) are caused by genetic defects that affect one or more lysosomal enzymes. These genetic diseases result generally from a deficiency in a particular enzyme activity present in the lysosome. To a lesser extent, these diseases may be due to deficiencies in proteins involved in lysosomal biogenesis. At the present, more than 40 distinct LSDs have been identified. Table A relates LSDs to the deficiency of their corresponding factor(s):
TABLE ALysosomal diseaseLysosomal enzyme involved1. Diseases related to lysosomal enzyme deficiencyHurler/Scheie syndrome or MPSα-L-iduronidasetype IGM1gangliosidosis, galactosialidosisβ-D-galactosidaseand Morquio syndrome B or MPS typeIVBGaucher diseaseβ-glucosidase (beta-glucocerebrosidase)Sandhoff diseaseβ-hexosaminidaseβ subunitTay-Sachs diseaseβ-hexosaminidaseα subunitβ-mannosidosisβ-D-mannosidaseα-L-fucosidosisα-L-fucosidaseMaroteaux-Lamy syndrome or MPSArylsulphatase Btype VIMetacromatic leukodystrophyArylsulphatase ASchindler diseaseα-N-acetylgalactosaminidaseAspartylglycosaminuriaAspartylglucosaminidaseHunter syndrome or MPS type IIIduronate-2-sulfataseSanfilippo syndrome A or MPS typeGlucosamine-N-sulfataseIIIASanfilippo syndrome B or MPS typeα-N-acetylglucosaminidaseIIIBSanfilippo syndrome C or MPS typeIIICAcetylcoenzymeA:α-glucosaminide-N-acetyltransferaseSanfilippo syndrome D or MPS type IIIDN-acetylglucosamine-6-sulfataseMorquio syndrome A or MPS type IVAN-acetylgalactosamine-6-sulfataseSly syndrome or MPS type VIIβ-D-glucuronidaseHyaluronidase deficiency orHyaluronidaseMPS type IXMultiple sulfatase deficiencyArylsulphatase A, B, Cα-mannosidosisα-L-mannosidaseSialidosisα-neuraminidaseX-linked ictiosis and multipleSteroid sulfatasesulfatase deficiencyMucolipidosis II and IIIPhosphotransferaseWolman diseaseAcid lipase, Tryoleil lipase,Cholesteryl esteraseFarber diseaseAcid ceramidaseNiemann-Pick disease type A and BSphingomyelinasePompe disease or glycogenosis typeα-glucosidaseIINeuronal ceroid lipofucsinosis,Palmitoyl proteininfantile typethioesteraseNeuronal ceroid lipofucsinosis,Carboxipeptidaselate infantile typeNeuronal ceroid lipofucsinosis,Lysosomal membrane proteinjuvenile typeKrabbe diseaseβ-galactocerebrosidaseLysosomal acid phosphataseAcid phosphatasedeficiencyPycnodysostosisCathepsine KLysosomal DiseaseTransporter protein involved2. Diseases related to lysosomal transporter protein deficiencyCystinosisCystine transporterSialic acid storage diseaseSialic acid transporterCobalamin deficiency type FCobalamin transporterNiemann-Pick disease type CNPC1 free-cholesteroltransporter proteinLysosomal DiseaseProtein involved3. Diseases related to lysosomal protective protein deficiencyGalactosialidosisNeuraminidase,β-galactosidase protectiveproteinLysosomal DiseaseFactor involved4. Diseases related to lysosomal enzyme activator deficiencyMetacromatic leukodystrophySaposin BvariantGaucher disease variantSaposin CTay-Sachs disease type ABβ-hexosaminidase activatorproteinGlycogen storage diseaseUnknown
LSDs are individually rare, although as a group these disorders are relatively common in the general population. The combined prevalence of LSDs is approximately 1 per 5,000 live births. See Meikle et al., JAMA, 281:249-254 (1999). Some groups within the population, as for example, descendants of Central and Eastern European (Ashkenazi) Jews are afflicted by a particularly high occurrence of LSDs. For instance, the prevalence rates in the Ashkenazi population of the Gaucher and Tay-Sachs diseases are 1 per 600 and 1 per 3,900 births, respectively.
The Hurler/Scheie syndrome or MPS type I is produced by the deficiency of the lysosomal enzyme α-L-iduronidase. The incidence of this disorder is about 1 in 111,000 births. See Meikle et al., supra. The frequencies for the Hurler and Scheie variants of this disorder are 1 in 100,000 and 1 in 600,000 births, respectively. The Hurler/Scheie syndrome produces a progressive degeneration of the brain, corneal opacities, enlargement of liver and spleen, an important bone dysostosis and a peculiar coarsening of facial features of the patient (Gargoyle face).
The cause of GM1 gangliosidosis is the deficiency of the lysosomal enzyme β-D-galactosidase. The occurrence of this disease is about 1 in 422,000 births. See Meikle et al., supra. GM1 gangliosidosis is a cerebral disease of infantile onset combining dysostosis multiplex, hepato-splenomegaly, cherry red macular spots and progressive neurologic deterioration.
The Gaucher disease is provoked by a deficiency of the lysosomal enzyme β-D-glucosidase. The incidence of this LSD is about 1 in 59,000 births for the general population. See Meikle et al., supra. However, the frequency of Gaucher disease type 1 (nonneuronopathic) in the Ashkenazi population is extremely high. About 1 in 600 births in this group show this particular LSD. Gaucher disease produces a severe hepato-splenomegaly, hematological disturbances, progressive involvement of bones and a severe neurological disease in a limited number of patients.
The chitotriosidase is a lysosomal enzyme that shows usually low activity in the serum and leukocytes of healthy people. However, in LSDs patients, especially in those with Gaucher disease, an increased activity of this enzyme is related with the progression of that disorder. See Young, et al., J. Inherit. Metab. Dis., 20(4): 595-602 (1997). Although the non-specificity of this enzyme limits its diagnostic capability, it is nonetheless, a good parameter to measure treatment effectiveness. See Den Tandt et al., Biochem. Mol. Med., 57:71-72 (1996). The biochemical diagnosis of chitotriosidase deficiency has been described and is known in the art.
Fabry disease is produced by the deficiency of the lysosomal enzyme α-D-galactosidase A. The incidence of Fabry disease is 1 in 117,000 births. See Meikle et al., supra. This LSD is characterized by distinctive skin lesions (angiokeratomes), periodic pain in the extremities, cerebrovascular and cardiovascular diseases and renal involvement.
The Sandhoff disease is caused by a deficiency in the activity of the lysosomal enzyme β-hexosaminidase β-subunit, which is a component of both hexosaminidase A and B. Individuals affected by this disorder show a low or non existent activity for both hexosaminidase A and B (total hexosaminidase). The incidence of this LSD is about 1 in 422,000 births. See Meikle et al., supra. This disorder produces a progressive cerebral degeneration starting at 6 months of age, blindness, hyperacusis and cherry red macular spots.
Tay-Sachs disease is produced by the deficiency of the lysosomal enzyme β-hexosaminidase α-subunit, which is a component of hexosaminidase A. Individuals afflicted by this disorder show a low or non existent activity of hexosaminidase A only. In the absence of hexosaminidase A, the GM2 ganglioside lipid accumulates abnormally in nervous cells causing progressive and irreparable damage to the brain. This LSD is a fatal genetic disorder that causes the degeneration of the central nervous tissue. The disorder is characterized by a progressive cerebral and retinal degeneration that starts in infancy, and is characterized with blindness, hyperacusis, cherry red macular spots and macrocephaly. The destructive process begins in the fetus during early pregnancy, although the disease is not clinically apparent until the child is several months old. The life expectancy of a child afflicted with classical Tay-Sachs disease is 5 years. See http://www.ntsad.org/, National Tay-Sachs & Allied Diseases Association (August 2000). The incidence of Tay-Sachs disease in the general population is approximately 1 in 222,000 births. See Meikle et al., supra. Its incidence is particularly high in descendants of Ashkenazi Jews. About 1 out of every 30 American Jews carries the Tay-Sachs gene. See http://www.ntsad.org/, supra.
The incidence of Mucolipidosis type II/III produced by the deficiency of the lysosomal enzyme N-acetylglucosaminyl-phosphotransferase is about 1 in 422,000 births. See Meikle et al., supra. The prevalence of this disorder is 1 in 325,000 and the estimated carrier frequency of 1 in 285. Both disorders are characterized by short stature, coarse features, joint rigidities, a progressive enlargement of liver and spleen, vertebral anomalies, and a variable degree of mental retardation. Mucolipidosis type II express the most severe phenotype. There is not therapy available for these disorders at the present. These LSDs are related to an increased level of some lysosomal enzymes such as total arylsulfatase (A and B). The abnormal lysosomal enzyme level is attributed to a defective post-translational modification as a consequence of phosphotransferase deficiency.
α-D-mannosidosis disease is provoked by the deficiency of the lysosomal enzyme α-D-mannosidase. The incidence of this disease is about 1 in 1,056,000 births. See Meikle et al., supra. α-D-mannosidosis patients develop severe mental and motor retardation, coarse features, hepato-splenomegaly, dysostosis multiplex, cataracts, corneal opacities and early death.
β-D-mannosidosis is provoked by the deficiency of the lysosomal enzyme β-D-mannosidase. The patients afflicted with this LSD show mental retardation, nerve deafness and angiokeratoma.
The α-L-fucosidosis is provoked by the deficiency of the lysosomal enzyme α-L-fucosidase. Patients afflicted with α-L-fucosidosis develop mental retardation, shortness of stature, coarse features, hepato-splenomegaly, dysostosis multiplex, increased sweet chloride and angiokeratomas.
Other LSDs are less known or has a low incidence in the general population. The biochemical diagnosis and characterization of LSDs has been widely reviewed and is known in the art. See Wenger et al., Techniques in Diagnostic Human Biochemical Genetics, A Laboratory Manual, pp. 587-617, F. A. Hommes Ed., Wiley-Liss, Inc. New York, N.Y. (1961); Practical Enzymology of the Sphingolipidoses, R. H. Glew and S. P. Peters, Eds., pp. 173-216, Alan R. Liss, Inc., NY (1977).
At the present most LSDs, such as α-D-mannosidosis, β-D-mannosidosis, α-L-fucosidosis and the Sandhoff and Tay-Sachs diseases, do not have an indicated therapy. Other LSDs, like for instance, late stage GM1 gangliosidosis, demand risky and costly therapeutical procedures such as bone marrow transplants.
Lately, recombinant proteins have been used for the treatment of some LSDs. Individuals afflicted with the Hurler/Scheie, Gaucher and Fabry diseases have been treated successfully with recombinant α-L-iduronidase, β-glucosidase and α-D-galactosidase, respectively. See Kakkis et al., Abstracts of the Joint Meeting of International Symposium on Innovative Therapies & 6th International Symposium on Mucopolysaccharidosis & Related Diseases, p. 23, May 19-21, Minneapolis, Minn., USA (2000); Grabowski et al., Blood Reviews, 12:115-133 (1998); Schiffmann, et al., J. Clin. Invest., 97:365-370 (2000). The application of recombinant proteins have shown promising result in the treatment of other LSDs, such as Pompe disease, now in Phase II clinical trials.
The positive effects of the known therapies, particularly for those LSDs involving central nervous system and bone pathologies, rely heavily on the early diagnosis and treatment of the disorder. A timely diagnosis may prevent the occurrence of irreversible damages in the patient. This consideration is especially true for LSDs where bone marrow transplant therapy is indicated. In these cases an early diagnosis of the LSD will allow clinicians to take advantage of the opportunity presented by the naturally suppressed immune system of the neonate thus enhancing the chances of a successful engraftment. See Ida et al., Abstracts of the Joint Meeting of International Symposium on Innovative Therapies & 6th International Symposium on Mucopolysaccharidosis & Related Diseases, May 19-21, Minneapolis, Minn., USA (2000).
Symptomatic LSDs patient show a high intralysosome storage of abnormal material. This situation is treated, when possible, by applying frequently high doses of recombinant enzymes to the LSD patient. At the present, this procedure entails administering intravenously the recombinant enzyme to the patient in order to assure that a sufficiently high level of enzyme will available in the body to degrade the abnormal material. If the LSD diagnosis is made in a pre-symptomatic stage, as for example, in newborns, the intralysosome storage of the abnormal material may be prevented. An early detection of these disorders will make possible to treat the LSD patient less frequently and with lower doses of recombinant enzyme. In addition, a reduced dosage treatment will also make possible to utilize less invasive routes of drug delivery like oral and nasal administration.
At the present, LSDs are diagnosed by DNA-based and enzymatic activity assays. Under DNA-based assays, a known mutation of a lysosomal enzyme gene is detected by hybridizing or sequencing part of the relevant enzymatic gene. See U.S. Pat. No. 5,710,028 (to Eyal et al.); U.S. Pat. No. 5,234,811 (to Beutler et al.); U.S. Pat. No. 5,217,865 (to Myerowitz). On the other hand, enzymatic activity assays implies measuring the amount of substrate or enzyme product related to a particular lysosomal enzyme by utilizing fluorogenic, spectrophotometric and radioactive analysis. See Wenger et al., supra.
For DNA-based assays, a specific region of DNA is amplified by a polymerase chain reaction. The amplified region is either sequenced entirely looking for mutations or hybridized with specific probes to detect a specific mutation in a lysosomal enzyme gene. However, the application of these techniques to LSD detection has been restricted by the following limitations:                1) In hybridization assays the test is limited to known mutations of the lysosomal enzyme under study. This method will not detect patients showing LSD symptoms if the LSD is caused by an unknown genetic mutation of the relevant lysosomal enzyme.        2) For sequencing assays the detection of a new mutation does not imply necessarily that the subject will develop the related LSD. For sequencing assays it is unfeasible to detect carrier individuals. Carrier detection must be done by hybridization assays.        
The lysosomal enzyme activity assays known in the art not based in DNA technology have also several restrictions in their application. One of their principal limitations is the minimum sample volume (usually between 5 to 10 ml of blood) necessary for testing. This volume is too high to allow infants to stand neonatal screening for these disorders. The consequences of this limitation are substantial. In most LSDs cases, a second diseased child is born to LSD carrier parents before the first born is identified as a LSD patient. The timely screening of the first born would prevent the development of the disorder and improve the chances of effective treatment for the rest of his siblings.
Another inconvenience of the enzymatic activity assays is that the sources of samples more commonly utilized for these tests (whole blood, plasma and serum) must be stored under controlled conditions and only for limited periods of time (no more than 3 days). These restrictions limit considerably the time that may lapse between the sample collection and the enzymatic activity assay.
Two additional limitations of the known lysosomal enzyme activity assays are their complexity and unavailability. Leukocyte isolation and purification from blood is a very specialized and tedious laboratory procedure. In addition, these assays are not performed in routinary clinical testing. These conditions restrict considerably the access of the general population to this type of tests. See Wenger et al., supra.
Recently, U.S. Pat. No. 5,719,035 (to Rosenthal et al.) have described a method for assaying enzyme activity in blood. This method discloses the determination of erythrocyte enzyme activities such as biotinidase and galactose-1-phosphate uridyl transferase (GALT). This method uses whole liquid blood and refers to the detection of non-lysosomal enzymes. The method revealed by Rosenthal also requires that hemoglobin be precipitated for testing.
A method for the diagnosis of LSDs using fluorophore assisted carbohydrate electrophoresis is also known in the art. See U.S. Pat. No. 5,205,917 (to Klock). However, this method does not provide a conclusive LSD diagnosis. The method is unable to identify the specific deficient lysosomal enzyme. In practice this method is complemented with other conventional enzyme activity assays for LSDs diagnosis because of its unreliability.
Singer et al. have described a method for determining the activity of lysosomal enzymes by using tears collected on filter paper and then storing immediately the collected samples in a buffer. The method was applied to the detection of Tay-Sachs and Fabry diseases in newborns. See Singer et al., Lancet 2:1116 (1973). However, this method is not used in routine clinical practice, especially for newborns. The method requires that tears be collected by applying strands of filter paper to the patient eyes. This procedure is extremely uncomfortable to patients, especially infants, and sample collection is difficult. In addition, a relative high volume of tears is required for testing. It is frequently necessary to repeat sampling when using his method because the amount of sample collected is insufficient for assay.
In addition, Hopwood et al. have disclosed a method for measuring a lysosome-associated membrane protein (LAMP-1) as a LSD diagnostic marker. See Hopwood et al., Clinical Chem., 45(8):1325-1335 (1997). The method attempts to determine indirectly the activity of lysosomal enzymes. However, this method fails to distinguish clearly between healthy and diseased individuals and has been found to be irreproducible in practice.